Ubiquitin-dependent proteolysis ensures that specific protein functions are turned off at the right time, in the right place and in a unidirectional fashion. The degradation of many cell cycle regulatory proteins is controlled by two classes of ubiquitin ligases: the SCF (Skp1-Cull-F-box protein) complexes and the Anaphase Promoting Complex/Cyclosome (APC/C). In humans there are sixty-eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin conjugating enzyme. Despite the large number of F-box proteins, only three human SCF ubiquitin ligases (SCFskp2, SCF?Trcp and SCFFbw7) have well- established functions and substrates, many of which are involved in cell cycle control (e.g., Cdc25A, cyclin E, Emi1, p21, p27 and Wee1). Given the crucial function of the cell cycle machinery, altered proteolysis of cell cycle regulators is clearly a contributing determinant of the unrestrained proliferation typical of cancer cells. Significantly, of the three characterized F-box proteins, Skp2 is the product of a proto-oncogene, Fbw7 is a tumor suppressor, and overexpression of ?Trcp contributes to transformation, at least in certain epithelial tissues. During the first nine years, CA76584 supported the elucidation of the molecular and cellular mechanisms by which three ubiquitin ligase complexes (SCFSkp2, SCF?Trcp and APC/CCdh1) control cell cycle progression through the degradation of cancer-relevant substrates that regulate the activity of CDKs. Furthermore, the corruption of these pathways occurring in cancer was revealed. Novel preliminary studies show that high levels of the F-box proteins Emi1 and Emi2 correlate with an increased stability of Skp2 in human cancers, and suggest that Emi1 and Emi2 function as oncoproteins. Based on these results, the new aims proposed under the third cycle of CA76584 are focused on a new tier of deregulation of the ubiquitin ligase/cell cycle network and its involvement in cancer: To determine whether the expression of Emil and Emi2 is deregulated in tumors and to investigate the mechanisms deregulating Skp2 stability in cancer cells (Aim 1);To study the contribution of Emi2 to cancer development using tissue culture systems and in vivo experiments (Aim 2);To study the cell cycle functions of Emi2 in cancer cells and to identify its biologically significant substrates (Aim 3). As the mechanisms of the ubiquitin-mediated proteolysis of cell cycle regulators are unraveled, this team is committed to the integration of its basic research results with an understanding of malignant transformation.